There is limited knowledge about the mechanisms by which GABA-A receptors are concentrated at inhibitory GABAergic Gray's type-2 synapses. The GABA-A receptors play a major role in brain function. It has been estimated that 40% of the brain synapses are GABAergic. The long-term objective of this proposal is to identify the proteins of the type-2 GABAergic synapses involved in the postsynaptic clustering and anchoring of GABA-A receptors. The aims of this proposal are:1) To demonstrate that GRIP proteins are involved in the postsynaptic clustering of gephyrin and GABA-A receptors; 2) To study the the relationship between the synaptic clustering of a protocadherin, whose intracellular domain interacts with the large intracellular loop of the gamma 2 subunit of the GABA-A receptor, and the synaptic clustering of GABA-A receptors and 3) To test the hypothesis that septins 6 and 7, which are enriched in type-2 postsynaptic densities in the rat brain, are involved in GABA-A receptor clustering and/or exocytosis. Light microscopy and electron microscopy immunocytochemistry with specific antibodies will be used for revealing the synaptic co-localization and clustering of GABA-A receptors and synaptic proteins in the intact brain and in hippocampal cultures. Identification of synaptic proteins will be done by mass spectrometry. Techniques such as siRNA treatment of cultured hippocampal neurons and the expression of tagged postsynaptic proteins in these cultures and in the intact brain will also be used to elucidate the synaptic targeting and clustering of the synaptic proteins and GABA-A receptors. These studies are relevant for understanding the molecular components of GABAergic synapses, their synaptic targeting and postsynaptic clustering as well as the mechanisms involved in neuronal connectivity and synaptic plasticity. It is predicted that the inappropriate synaptic and extrasynaptic localization of GABA-A receptors affects GABAergic synaptic function and brain development, leading to epilepsy and other neurological and mental disorders.